Noninvasive intraductal fluid diagnostic screen

ABSTRACT

Disclosed is a method and apparatus for accomplishing a noninvasive screen for breast disease markers, including breast cancer markers and cytologically abnormal cells. Intraductal fluid is noninvasively aspirated using compression, heating and suction cycles. The removed sample is thereafter assayed for the presence of cytologically abnormal cells and/or one or more breast disease markers. Sample size and intraductal mobility of breast disease markers may be enhanced by retrograde introduction of a carrier. Devices and assays are also disclosed.

[0001] The present invention relates to noninvasive screening assays forindicium of breast cancer or other breast disease.

BACKGROUND OF THE INVENTION

[0002] Breast cancer is by far the most common form of cancer in women,and is the second leading cause of cancer death in humans. Despite manyrecent advances in diagnosing and treating breast cancer, the prevalenceof this disease has been steadily rising at a rate of about 1% per yearsince 1940. Today, the likelihood that a woman living in North Americawill develop breast cancer during her lifetime is one in eight. Thecurrent widespread use of mammography has resulted in improved detectionof breast cancer. Nonetheless, the death rate due to breast cancer hasremained unchanged at about 27 deaths per 100,000 women. All too often,breast cancer is discovered at a stage that is too far advanced, whentherapeutic options and survival rates are severely limited.Accordingly, more sensitive and reliable methods are needed to detectsmall (less than 2 cm diameter), early stage, in situ carcinomas of thebreast. Such methods should significantly improve breast cancersurvival, as suggested by the successful employment of Papinicolousmears for early detection and treatment of cervical cancer.

[0003] In addition to the problem of early detection, there remainserious problems in distinguishing between malignant and benign breastdisease, in staging known breast cancers, and in differentiating betweendifferent types of breast cancers (e.g. estrogen dependent versusnon-estrogen dependent tumors). Recent efforts to develop improvedmethods for breast cancer detection, staging and classification havefocused on a promising array of so-called cancer “markers.” Cancermarkers are typically proteins that are uniquely expressed (e.g. as acell surface or secreted protein) by cancerous cells, or are expressedat measurably increased or decreased levels by cancerous cells comparedto normal cells. Other cancer markers can include specific DNA or RNAsequences marking deleterious genetic changes or alterations in thepatterns or levels of gene expression associated with particular formsof cancer.

[0004] A large number and variety of breast cancer markers have beenidentified to date, and many of these have been shown to have importantvalue for determining prognostic and/or treatment-related variables.Prognostic variables are those variables that serve to predict diseaseoutcome, such as the likelihood or timing of relapse or survival.Treatment-related variables predict the likelihood of success or failureof a given therapeutic plan. Certain breast cancer markers clearly serveboth functions. For example, estrogen receptor levels are predictive ofrelapse and survival for breast cancer patients, independent oftreatment, and are also predictive of responsiveness to endocrinetherapy. Pertschuk et al., Cancer 66: 1663-1670, 1990; Parl and Posey,Hum. Pathol. 19: 960-966, 1988; Kinsel et al., Cancer Res. 49:1052-1056, 1989; Anderson and Poulson Cancer 65: 1901-1908, 1989.

[0005] The utility of specific breast cancer markers for screening anddiagnosis, staging and classification, monitoring and/or therapypurposes depends on the nature and activity of the marker in question.For general reviews of breast cancer markers, see Porter-Jordan et al.,Hematol. Oncol. Clin. North Amer. 8: 73-100, 1994; and Greiner,Pharmaceutical Tech., May, 1993, pp. 28-44. As reflected in thesereviews, a primary focus for developing breast cancer markers hascentered on the overlapping areas of tumorigenesis, tumor growth andcancer invasion. Tumorigenesis and tumor growth can be assessed using avariety of cell proliferation markers (for example Ki67, cyclin D1 andproliferating cell nuclear antigen (PCNA)), some of which may beimportant oncogenes as well. Tumor growth can also be evaluated using avariety of growth factor and hormone markers (for example estrogen,epidermal growth factor (EGF), erbB-2, transforming growth factor(TGF).alpha.), which may be overexpressed, underexpressed or exhibitaltered activity in cancer cells. By the same token, receptors ofautocrine or exocrine growth factors and hormones (for example insulingrowth factor (IGF) receptors, and EGF receptor) may also exhibitchanges in expression or activity associated with tumor growth. Lastly,tumor growth is supported by angiogenesis involving the elaboration andgrowth of new blood vessels and the concomitant expression of angiogenicfactors that can serve as markers for tumorigenesis and tumor growth.

[0006] In addition to tumorigenic, proliferation and growth markers, anumber of markers have been identified that can serve as indicators ofinvasiveness and/or metastatic potential in a population of cancercells. These markers generally reflect altered interactions betweencancer cells and their surrounding microenvironment. For example, whencancer cells invade or metastasize, detectable changes may occur in theexpression or activity of cell adhesion or motility factors, examples ofwhich include the cancer markers Cathepsin D, plasminogen activators,collagenases and other factors. In addition, decreased expression oroverexpression of several putative tumor “suppressor” genes (for examplenm23, p53 and rb) has been directly associated with increased metastaticpotential or deregulation of growth predictive of poor disease outcome.

[0007] Thus, the evaluation of proliferation markers, oncogenes, growthfactors and growth factor receptors, angiogenic factors, proteases,adhesion factors and tumor suppressor genes, among other cancer markers,can provide important information concerning the risk, presence, statusor future behavior of cancer in a patient. Determining the presence orlevel of expression or activity of one or more of these cancer markerscan aid in the differential diagnosis of patients with uncertainclinical abnormalities, for example by distinguishing malignant frombenign abnormalities. Furthermore, in patients presenting withestablished malignancy, cancer markers can be useful to predict the riskof future relapse, or the likelihood of response in a particular patientto a selected therapeutic course. Even more specific information can beobtained by analyzing highly specific cancer markers, or combinations ofmarkers, which may predict responsiveness of a patient to specific drugsor treatment options.

[0008] Methods for detecting and measuring cancer markers have beenrevolutionized by the development of immunological assays, particularlyby assays that utilize monoclonal antibody technology. Previously, manycancer markers could only be detected or measured using conventionalbiochemical assay methods, which generally require large test samplesand are therefore unsuitable in most clinical applications. In contrast,modern immunoassay techniques can detect and measure cancer markers inrelatively much smaller samples, particularly when monoclonal antibodiesthat specifically recognize a targeted marker protein are used.Accordingly, it is now routine to assay for the presence or absence,level, or activity of selected cancer markers by immunohistochemicallystaining breast tissue specimens obtained via conventional biopsymethods. Because of the highly sensitive nature of immunohistochemicalstaining, these methods have also been successfully employed to detectand measure cancer markers in smaller, needle biopsy specimens whichrequire less invasive sample gathering procedures compared toconventional biopsy specimens. In addition, other immunological methodshave been developed and are now well known in the art which allow fordetection and measurement of cancer markers in non-cellular samples suchas serum and other biological fluids from patients. The use of thesealternative sample sources substantially reduces the morbidity and costsof assays compared to procedures employing conventional biopsy samples,which allows for application of cancer marker assays in early screeningand low risk monitoring programs where invasive biopsy procedures arenot indicated.

[0009] For the purpose of breast cancer evaluation, the use ofconventional or needle biopsy samples for cancer marker assays is oftenundesirable, because a primary goal of such assays is to detect thecancer before it progresses to a palpable or mammographically detectabletumor stage. Prior to this stage, biopsies are generallycontraindicated, making early screening and low risk monitoringprocedures employing such samples untenable. Therefore, there is generalneed in the art to obtain samples for breast cancer marker assays byless invasive means than biopsy.

[0010] Thus, serum withdrawal has been attempted for breast cancermarker assays. Efforts to utilize serum samples for breast cancer markerassays have met with limited success. The targeted markers are eithernot detectable in serum, or telltale changes in the levels or activityof the markers cannot be monitored in serum. In addition, the presenceof breast cancer markers in serum may occur at the time ofmicro-metastasis, making serum assays less useful for detectingpre-metastatic disease. In contrast, fluid within the mammary glandsthemselves is expected to contain much higher and more biologicallyrelevant levels of breast cancer markers than serum, particularly inview of the fact that 80%-90% of all breast cancers occur within theintraductal epithelium of these glands. Fluid within the breast ducts isexpected to contain an assemblage and concentration of hormones, growthfactors and other potential markers comparable to those secreted by, oracting upon, the surrounding cells of the alveolar-ductal system.Likewise, mammary fluid is expected to contain cells and solid cellulardebris or products that can be used in cytological or immunologicalassays to evaluate intracellular or cell surface markers that may not bedetectable in the liquid fraction of mammary fluid.

[0011] Previous attempts to develop non-invasive breast cancer markerassays utilizing mammary fluid samples have included studies of mammaryfluid obtained from patients presenting with spontaneous nippledischarge. In one of these studies, conducted by Inaji et al., Cancer60: 3008-3013, 1987, levels of the breast cancer marker carcinoembryonicantigen (CEA) were measured using conventional, enzyme linkedimmunoassay (ELISA) and sandwich-type, monoclonal immunoassay methods.These methods successfully and reproducibly demonstrated that CEA levelsin spontaneously discharged mammary fluid provide a sensitive indicatorof nonpalpable breast cancer. In a subsequent study, also by Inaji etal., Jpn. J. Clin. Oncol. 19: 373-379, 1989, these results were expandedusing a more sensitive, dry chemistry, dot-immunobinding assay for CEAdetermination. This latter study reported that elevated CEA levelsoccurred in 43% of patients tested with palpable breast tumors, and in73% of patients tested with nonpalpable breast tumors. CEA levels in thedischarged mammary fluid were highly correlated with intratumoral CEAlevels, indicating that the level of CEA expression by breast cancercells is closely reflected in the mammary fluid CEA content. Based onthese results, the authors concluded that immunoassays for CEA inspontaneously discharged mammary fluid are useful for screeningnonpalpable breast cancer.

[0012] Although the evaluation of mammary fluid has been shown to be auseful method for screening nonpalpable breast cancer in women whoexperience spontaneous nipple discharge, the rarity of this conditionrenders the methods of Inaji et al, inapplicable to the majority ofwomen who are candidates for early breast cancer screening. In addition,the first Inaji report cited above determined that certain patientssuffering spontaneous nipple discharge secrete less than 10 μl ofmammary fluid, which is a critically low level for the ELISA andsandwich immunoassays employed in that study. It is likely that otherantibodies used to assay other cancer markers may exhibit even lowersensitivity than the anti-CEA antibodies used by Inaji and coworkers,and may therefore not be adaptable or sensitive enough to be employedeven in dry chemical immunoassays of small samples of spontaneouslydischarged mammary fluid.

[0013] In view of the above, an important need remains in the art formore widely applicable, non-invasive methods and devices for obtainingbiological samples for use in evaluating, diagnosing and managing breastdisease including cancer, particularly for screening early stage,nonpalpable breast tumors. Biological samples thus obtained can be usedto evaluate, diagnose and manage breast disease, particularly bydetecting or measuring selected breast cancer markers, or panels ofbreast cancer markers, to provide highly specific, cancer prognosticand/or treatment-related information, and to diagnose and managepre-cancerous conditions, cancer susceptibility, breast infections andother breast diseases.

SUMMARY OF THE INVENTION

[0014] There is provided in accordance with one aspect of the presentinvention, a method of screening intraductal breast fluid for one ormore breast disease markers. The method comprises the steps ofcontacting the breast with a mechanical intraductal fluid aspirationdevice, activating the device to apply peristaltic compression andsuction to the breast during a period of nonlactation to removeintraductal breast fluid, and screening the fluid for breast diseasemarkers. The method preferably further comprises the step of applyingheat from the device to the breast.

[0015] In accordance with another aspect of the present invention, thereis provided an intraductal breast fluid screening device. The devicecomprises a tissue-contacting surface defining a first concavity forreceiving a breast and a second concavity for receiving a nipple. Adriver is provided for imparting compressive force on at least a portionof the tissue contacting surface defining the first concavity. A vacuumconduit is in communication with the second concavity, and a samplecollector is in communication with the second concavity. Preferably, thesample collector comprises a collection patch, which may include aspecific binding partner for an analyte of interest.

[0016] In one embodiment, the driver imparts peristaltic compressiveforce on the tissue contacting surface. For this purpose, the drivercomprises at least one expandable chamber, which may be filled oremptied to impart compressive force.

[0017] In accordance with a further aspect of the present invention,there is provided an intraductal fluid aspiration device. The devicecomprises a control unit, and a power head. A flexible control lineconnects the power head to the control unit. A disposable user interfaceis removably attached to the power head. A vacuum source in the controlunit is in communication with the user interface through the controlline. A heat source is placed in thermal communication with the userinterface, and a compression cycle generator is in force transmittingcontact with the user interface. Preferably, a sample collector is influid communication with the user interface. In one embodiment, thesample collector is removable from the user interface.

[0018] In accordance with a further aspect of the present invention,there is provided a method of increasing yield in a breast ductalaspiration. The method comprises the steps of providing a carrier, andintroducing the carrier under pressure retrograde into the duct. Thecarrier is thereafter recovered from the duct, using a combination ofcompression, heat, and suction. Samples found in the recovered carriermay thereafter be examined such as by cytological examination orbiochemical assay.

[0019] Further features and advantages of the present invention willbecome apparent to those of skill in the art in view of the detaileddescription of preferred embodiments which follows, when consideredtogether with the attached drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is a schematic illustration of a portable, self-containedintraductal fluid aspiration device.

[0021]FIG. 2 is an illustration of a portable self-contained intraductalfluid aspiration device as in FIG. 1, schematically illustrating aplurality of annular compression rings.

[0022]FIG. 3 is a schematic illustration of a desktop embodiment of anintraductal fluid aspiration device in accordance with the presentinvention.

[0023]FIG. 4 is a schematic illustration of a sample collection patch,in communication with the flow path between the patient and a vacuumsource.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0024] Referring to FIG. 1, there is illustrated a schematicrepresentation of a portable, self-contained intraductal fluidaspiration device 20 in accordance with one aspect of the presentinvention. The aspiration device 20 includes a housing 22, forcontaining various controls and functional components of the device 20.One or more controls and/or indicators 25 may be provided on thehousing, for controlling various aspects of the device such as suction,compression, and other features (e.g., heat, ultrasound) which may beincluded depending upon the intended functionality of the aspirationdevice 20. The housing 22 may be formed by extrusion, injection moldingor other well known techniques from a suitable biocompatible materialsuch as high density polyethylene, nylon, polyethylene terephthalate, orothers well known in the art. The housing is preferably formed in anergonomic configuration, to comfortably facilitate grasping in one handduring use.

[0025] The housing 22 is provided with a patient or breast interface 24,which may either be permanently attached to the housing 22 or removablyattached such as for cleaning or disposal. Breast interface 24 has aproximal end 26, a distal end 28, and a body 30 extending therebetween.The interface 24 has a tissue contacting surface 32 defining a firstconcavity 34 for receiving a breast and a second concavity 38 forreceiving a nipple. The tissue contacting surface 32 may be an integralsurface on the body 30, or may comprise a separate interior liner whichis adhered to or otherwise fit within and/or secured to the body 30.

[0026] The body 30 may be manufactured in any of a variety of ways, suchas injection molding, blow molding tube stock within a tapered capturetube, or other known manners, using any of a variety of well knownbiocompatible polymeric materials. Preferably, the body 30 istransparent, which may be achieved by forming from polycarbonate, orother relatively clear materials known in the art. In one embodiment,the generally frusto-conical body 30 is sufficiently rigid to providesupport for a flexible interior liner.

[0027] The dimensions of the interface 24 may be varied widely, as willbe appreciated by those of skill in the art in view of the disclosureherein. In general, the distal end 28 of the flexible body 30 isprovided with an elastic sealing ring 35 having an inside diameterwithin the range of from about 2″ to about 10″. The distal limit of thesecond concavity 38 has an inside diameter within the range of fromabout 1″ to about 4″. The first concavity 34 has an axial length fromproximal end 26 to distal end 28 within the range of from about 0″ toabout 12″, and, in many embodiments, within the range of from about 2″to about 6″. The first concavity 34 has a generally conical or bellshaped interior configuration, as will be appreciated by those of skillin the art.

[0028] Preferably, the breast interface 24 is provided with a dynamiccompression zone 42, having one or more compression elements 45 forcompression in the mid breast region to facilitate intraductal fluidaspiration. Although the specific dimensions will vary from patient topatient, as well as with age and parity, the breast includes a pluralityof ducts which are generally confluent in the direction of a pluralityof external openings on the nipple. Most of the intraductal volume iscontained in the distal one-half or one-third of the breast (from thepatient's perspective). Thus, the inventors presently believe that acompression zone approximately centered around the midbreast region andextending anatomically distally will optimize fluid transport in theduct.

[0029] Referring to FIG. 2, the dynamic compression zone 42 isschematically illustrated (not to scale) as comprising a plurality ofannular compression rings 44, 46, 48 and 50. Preferably, the annularcompression rings are in operative communication with a driver 52 in thehousing 22, to permit sequential operation. The preferred operation modemimics a peristaltic motion such that tissue compression is accomplishedsequentially proximally with respect to the device starting withcompression ring 44 followed by compression ring 46 followed bycompression ring 48 followed by compression ring 50. As will be apparentto those of skill in the art in view of the disclosure herein, any of awide variety of compression ring numbers and configurations may beutilized in accordance with the present invention. Thus, theillustration of four compression rings in FIG. 2 is not consideredlimiting on the scope of the invention. In general, anywhere from aboutone to about twenty compression elements 45 may be utilized, in ringform or nonannular form, and, preferably, between about three and tenare contemplated in most embodiments.

[0030] The compression elements 45 may comprise any of a variety ofstructures, such as inflatable tubular elements or other inflatablestructures, or mechanical compression elements such as rollers. In theillustrated embodiment, which is not drawn to scale in order to improveclarity, the dynamic compression zone 42 comprises a plurality ofannular, inflatable, tubular compression rings each of which isconnected to the driver 52 by a unique conduit 56. The driver 52preferably includes a microprocessor or other central processing unitfor sequentially driving the compression elements 45 as describedpreviously. In one embodiment, the driver 52 includes a pump forcontrollably inflating and deflating each compression ring in responseto the microprocessor and in accordance with the predeterminedcompression protocol. Inflation media such as air, water, or gel may beutilized, depending upon the desired performance characteristics. In oneembodiment, a heat retaining gel such as morphing gel, available fromDow Corning, is utilized to enable the delivery of heat during thecompression cycle.

[0031] The compression elements 45 may alternatively be connected toeach other by a capillary tube or flow restriction orifice, or presssurerelief valves to enable compression (inflation) in a predeterminedsequence. Alternatively, the compression elements 45 may be in fluidcommunication with each other, with each having a wall with a uniquedurometer or elasticity such that each element inflates as a uniquethreshold inflation pressure is reached and/or exceeded.

[0032] The microprocessor may be programmed to a particular pumping andcompression cycle characteristic, or may be adjustable by the user tooptimize the aspiration function as desired. For example, compressioncycles may be peristaltic, with a sequential compression pattern fromchest wall (distal end 28) to the proximal end 26. Alternatively, thecompression cycle may be non peristaltic pulsitile. Vacuum may beapplied constantly throughout the pumping cycle, or may be pulsitileeither in phase or out of phase with the compression cycles.

[0033] The aspiration device 20 is further provided with a vacuumgenerator such as a pump in the housing 22, in communication with thesecond concavity 38 by way of a vacuum conduit (not shown). Associatedelectronics, such as a power source and driving circuitry are preferablyconnected to a control 25 to enable the user to selectively activate anddeactivate the vacuum. Alternatively, the pump and vacuum functions maybe fully automatic, and pre-programmed into the micro-processor. Thepump is generally capable of generating a vacuum within an operatingrange of from 0 (pump off) to about 300 mm/Hg. Although vacuum in excessof 300 mm/Hg may also be utilized, vacuum in this area or higher maycause rupture of microvasculature and is unnecessary to accomplish theobjectives of the present invention. For this reason, limit valves maybe provided in communication with the vacuum conduit, as are known inthe art, to limit the vacuum to no more than about 200 mm/Hg, or 250mm/Hg, or 300 mm/Hg. Within the methods of the invention, negativepressures of 50-200 mm Hg are preferred, and these pressures aremaintained, preferably intermittently, for approximately 1-15 minutes,depending on the sensitivity of individual patients, oxytocin dosage andother factors.

[0034] The foregoing embodiment is useful in a variety of settings,particularly for in home intraductal aspiration. In an alternativeembodiment of the present invention, a desk top unit 60 is provided,such as for the physician's office or other conventional clinicalsetting. See FIG. 3. The desk top intraductal fluid aspiration system 60comprises a control unit 62, in communication with a power head 64 byway of an elongate flexible control line 66. The power head 64 isprovided with a disposable user interface 68 which may be similar oridentical to the interface 24 described previously. In this embodiment,the interface 68 is preferably removably connected to the power head 64,to facilitate one time use and subsequent disposal of the interface 68.Alternatively, the entire interface and power head assembly may be onetime use disposable.

[0035] The control 62 preferably includes the vacuum pump, and otherdriver circuitry and controls as may be needed depending upon theintended functionality of the desk top unit 60. For example, a vacuumpump (not illustrated) is in communication with the disposable userinterface 68 by way of a vacuum lumen (not illustrated) extendingthroughout the length of the control line 66. Additional lumens orwiring extend through the control line 66 for accomplishing theperistaltic or other sequential compression motion of the dynamiccompression zone 42 as has been discussed.

[0036] In either a diagnostic or non-diagnostic embodiment, a samplecollector or reservoir is preferably positioned in fluid communicationwith the disposable user interface 68, to allow collection ofintraductal fluid. The sample collector or container may be removable,such as to enable transport of collected intraductal fluid to adiagnostic laboratory or other facility for diagnostic analysis.

[0037] Preferably, the disposable user interface 68 is provided with aheat source, such as a heat retaining gel or other media for surroundingor contacting the interface 24, and/or for inflating the compressionelements as has been previously discussed. Alternatively, resistanceheating elements may be provided in the disposable user interface 68 orassociated power head 64, powered by way of electrical conductorsextending throughout the control line 66. In an embodiment where thedynamic compression zone 42 includes elements filled with a heatretaining gel or other media for retaining heat, the breast interface 24may be removed and heated such as in a microwave oven or other heatsource prior to use. An ultrasound source may also be provided in thecontrol unit 62 or power head 64, for driving one or more ultrasoundtransducers in the power head 64 to assist in initial removal of keratinplugs that may occur at the opening of the ducts, and possibly also toserve as a heating source. Alternatively, a heating fluid may becirculated through a closed loop such as from a heater in the controlunit 62, through a first lumen in control line 66 to a heat exchanger inthe power head 64 or patient interface, and back through a second lumenin control line 66 to the control unit 62.

[0038] The volume of expressed mammary fluid will vary depending on avariety of factors, including patient sensitivity to oxytocin, if used,dosage of oxytocin delivered, time and pressure and other variables ofbreast pump administration, and other factors. For certain relativelylow sensitivity breast marker assays, a volume of expressed mammaryfluid of 300-500 μl is preferred to provide ample material forconducting the assay, and these volumes are expected to be obtainablefrom a substantial proportion of women treated according to the abovemethods. To express 300-500 μl of mammary fluid, some women will requirerepeated stimulation treatments, perhaps requiring pooling of mammaryfluid samples obtained during multiple patient visits. However, for moresensitive assays of the invention, e.g. solid phase immunoassays, muchsmaller samples of 3 μl or less may be sufficient to carry out theassays. This is particularly so in the case of breast cancer markersthat are naturally secreted into the mammary fluid and are thereforeexpected to be present in very high concentrations compared to, forexample, breast epithelial cell surface antigens or intracellularantigens that may not be secreted.

[0039] Although one aspect of the present invention lies in the novel,non-invasive methods for obtaining biological samples from mammaryfluid, an additional aspect of the invention involves the use of thecollected sample for detecting and/or measuring important breast diseasemarkers. The invention thus enables the convenient application of abroad range of assay methods incorporating known procedures and reagentsfor determining the presence and/or expression levels of breast diseasemarkers, particularly breast cancer markers, in biological samples.

[0040] During or after the mammary fluid expression step, a biologicalsample is collected from the expressed mammary fluid. A range ofsuitable biological samples are contemplated and will be useful withinthe methods of the invention, including whole mammary fluid, selectedliquid or solid fractions of the mammary fluid, whole cells or cellularconstituents, proteins, glycoproteins, peptides, nucleotides (includingDNA and RNA polynucleotides) and other like biochemical and molecularconstituents of the mammary fluid.

[0041] Sample collection can be achieved simply by receiving theexpressed mammary fluid within a suitable reservoir such as within or incommunication with the convavity 38 with or without an absorptive samplecollection media. Samples can be collected directly on to orsubsequently exposed to conventional buffers, diluents, extraction orchromatographic media, filters, etc., to stabilize or prepare the samplefor further processing or direct incorporation into a desired assay. Incertain embodiments of the invention, the expressed mammary fluid iscollected directly onto a solid phase medium, such as a microscopicglass slide, nitrocellulose filter, affinity column, dot blot matrix orother like medium, that will selectively adsorb, bind, filter orotherwise process desired components of the mammary fluid, such as bulkor selected proteins, for convenient incorporation into a desired assay.

[0042] Thus, referring to FIG. 4, a flow path such as lumen 39 drawscollected fluid from the patient interface 24 through a samplecollection patch 41. The sample collection patch 41 may be positioneddirectly against the external opening of the ducts, to minimize fluidloss in the device.

[0043] In the illustrated embodiment, the sample collection patch 41 ismoveably positioned within the flow path, with a mild biasing force inthe distal direction. In this manner, the patch 41 can maintain lowpressure contact with the distal surface of the nipple throughout arange of axial positions along the longitudinal axis of the patientinterface. Preferably, an axial range of motion is provided for at leastthe tissue contacting portion of the patch 41 of at least about 0.25inches. In some embodiments, the range of from about 0.5 inches to about2 inches or more may be accomplished. In the illustrated embodiment, theaxial motion of the sample collection patch 41 is achieved by allowingbending or pivoting of the patch 41, throughout the patch and/or at areleasable attachment point 43 to either the patient interface 24 or thehousing 22. Axial movement of the sample collection patch mayalternatively be accomplished by mounting the sample collection patch onthe surface of a compressible foam, which will compress in response topressure from the patient. Alternatively, the compressible foam may formthe sample collection media, without a separate patch, as is describedbelow.

[0044] The sample collection patch 41 or other sample collection mediamay be removably attached to the aspiration device 20 such as by one ormore releasable connections 47, which may comprise adhesive surfaces ormechanical interfit surfaces such as an annular recess for receiving thepatch 41, radially inwardly extending tabs for receiving the patch 41,or others as will be apparent to those of skill in the art in view ofthe disclosure herein. The patch 41 may consist entirely of a flexibleabsorbtive medium. Alternatively, the patch may include an absorbtivemedium in combination with a support structure such as a backing plate,or an annular ring for surrounding the patch 41, and facilitatingreleasable connection to the aspiration device 20.

[0045] The patch 41 is preferably removable from the aspiration device20. The patch 41 may be removed through the first concavity or chamber34, by hand or using tweezers, hemostats or other retrieval device.Alternatively, the patch 41 may be removed through a lateral opening 49in the side wall of the aspiration device 20. As a further alternative,the patch 41 may be removed following disconnection of the patientinterface 24 from the housing 22.

[0046] The patch 41 or distal surface of a solid sample collectionmedium such as a foam may have any of a variety of configurations,depending upon other aspects of the device design. In the illustratedembodiment, the patch 41 is a generally planar membrane. Alternatively,the patch 41 or patient contacting surface of a solid foam or othercollection device may be conical or otherwise concave in the directionof the patient. The sample collection patch 41 or other sample collationstructure may also have a component which extends distally towards thepatient, from a lower portion of the patch 41, to capture any samplewhich may drop under the influence of gravity without first beingabsorbed by the patch 41.

[0047] The fluid capacity of the patch 41 or other collection media maybe varied, depending upon the intended patient population and purpose ofthe aspiration. In general, sample sizes in the microliter or lowmilliliter range may be useful for different types of assays. For acertain patient populations, the volume of fluid expressed may reach inexcess of 1 to 5 milliliter or greater, in which case a fluid collectionchamber may be provided on the device in the area of the illustratedopening 49 on FIG. 4. Any of a variety of such variations will beapparent to those of skill in the art in view of the disclosure herein,taking into account the purpose of the aspiration.

[0048] The composition and construction of sample collection patch 41will vary, depending upon the nature of the intended assay. For example,any of a wide variety of porous or absorptive materials may be utilizedto collect cellular and cellular component samples which may be used forcytological exam. Materials such as conventional filter paper, cottongauze, fiber webs such as knitted fabrics or nonwoven rayon or cellulosefibers may be used. A variety of microporous films comprising materialssuch as nylon 66, polycarbonate, modified polyvinyl fluoride andpolysulphone may alternatively be used. Embodiments of the collectionpatch 41 which are intended to permit chemical or biochemical assays mayadditionally be provided with any of a variety of binders for eitherchemically binding with an analyte or adsorbing the analyte to bedetermined. The binder layer may additionally include a specific bindingpartner of the analyte to be determined, such as a polyclonal ormonoclonal antibody or an antigen matched to a specific antibody desiredto be measured in the extracted fluid. Other binding systems which arematched to the desired analyte or analytes may be readily adapted foruse in the present invention, as will be understood by those of skill inthe art. The range of contemplated sample collection procedures andmaterials that are useful within the invention is broad, and selectedmethods and materials will vary with each selected assay, as will beunderstood and readily practiced by those skilled in the art.

[0049] The sample collection patch may be constructed of any suitablematerial. In preferred embodiments, the sample collection patch includesa membrane or filtration medium upon, through, or in which a fluidsample is collected. The sample collection patch may be of any suitableshape. While circular or square patches are preferred for manyapplications, other shapes may also be used. The sample collection patchshould be of sufficient size such that an adequate sample may becollected.

[0050] The sample collection patch may include a material capable ofproviding depth filtration or sieve filtration of a sample. In depthfiltration, particulates are trapped both within the matrix and on thesurface of the filtration medium. Depth filters are composed of randommats of metallic, polymeric, inorganic, or organic materials. Depthfilters rely on the density and thickness of the mats to trapparticulates, and generally retain large quantities of particulateswithin the matrices. Certain disadvantages of depth filters includemedia migration, which is the shifting of the filter medium understress, and particulate unloading at high differential pressures.Advantages of depth filters include reduced cost, high throughputs, highparticulate-holding capacity, removal of a range of particle sizes, andhigh flow rates.

[0051] In sieve filtration, particulates larger than the pore size of amembrane are trapped, while smaller particulates pass through themembrane but may be captured within the membrane by some othermechanism. Sieve filtration membranes are generally polymeric filmsapproximately 120 microns thick with a narrow pore size distribution.Certain disadvantages of sieve filtration include lower flow rates andlower particulate holding capacity. Advantages include absolutesubmicron pore size ratings, no channeling or bypass, capacity forretaining bacteria and particles, low extractables, sterilizable, andintegrity testable.

[0052] In various embodiments, the sample collection patch includes oneor more filtration media such as the filtration media manufactured byPall Gelman Sciences of East Hills, N.Y. Such filtration media that maybe suitable for use in particular assays of the various embodimentsinclude filtration media originally developed for use in bloodseparations. Such filtration media may include polyester filtrationmedia such as Pall Gelman's Hemasep™ modified polyester materials,polyether sulfone membranes such as the Presense™ membrane, Cytosep®single layer fiber composite membrane, and Leukosorb® medium. Othersuitable filtration media may include the Predator™ membrane, apolyether sulfone membrane that is surface modified to possess nitrogroups.

[0053] Pall Gelman's Biodyne® nylon 6,6 membranes may also be suitablefor use in sample collection patches for certain assays. UnmodifiedBiodyne® nylon 6,6 membrane may be preferred for some applications.Alternatively, the Biodyne® nylon 6,6 membrane may be surface modifiedwith quaternary ammonium groups so as to impart a positive charge to thepore surfaces, thereby promoting strong ionic bonding of negativelycharged analytes. Likewise, the Biodyne® nylon 6,6 membrane may besurface modified with carboxyl groups so as to impart a negative chargeto the pore surfaces, thereby promoting strong ionic bonding ofpositively charged analytes. Such carboxyl group surface-modifiedBiodyne® nylon 6,6 membranes may be derivatized via coupling reactionsthrough the carboxyl groups at the pore surfaces.

[0054] In various embodiments other membranes may be preferred for usein sample collection patches, such as Biotrace® nitrocellulosemembranes, Fluorotrans® polyvinylidene difluoride (PVDF) membranes,Immunodyne® ABC nylon 6,6 affinity membranes having a high density ofcovalent binding sites capable of permanently immobilizing proteins andpeptides on contact, and Ultrabind™ aldehyde-modified polyether sulfonemembranes capable of providing covalent binding to amine groups onproteins.

[0055] Various absorbent materials also available from Pall Gelman mayalso be used in certain embodiments, such as conjugate pads comprised ofborosilicate glass with no binder or with polyvinylacetate (PVA) oranother suitable binder, Loprosorb™ low protein binding hydrophilicfibrous medium, cellulose absorbent papers, Loprodyne® internallysupported nylon 6,6 membrane with low protein binding, or Z-Bind™post-treated modified polyethersulfone membrane.

[0056] Ion exchange membranes may be preferred for use in samplecollection patches for certain assays. Such membranes may include PallGelman's Raipore™ ion-exchange polytetrafluoroethylene (PTFE) cationicor anionic membranes, and microporous ion exchange membranes constructedof polyether sulfone and possessing either sulfonic acid or quaternaryammonium groups on the membrane surface.

[0057] In various applications, it may be preferred to use a hydrophobicand/or oleophobic material in a sample collection patch. Materialssuitable for use in such applications may include Pall Gelman'sHydrolon® nylon 6,6 membranes, Hydrolon® PTFE membranes, Supor® Rpolyethersulfone membrane, and Pallflex composite materials.

[0058] The above-mentioned filtration media available from Pall Gelmanare representative examples of the wide variety of filtration mediacommercially available that may be suitable for use in sample collectionpatches. Various filtration media available from other manufacturers mayalso be suitable for use in sample collection patches, as maycustom-manufactured filtration media. Suitable filtration media mayinclude a single material, e.g., a single membrane, or may be acomposite manufactured from two or more materials, e.g., variouscombinations of membranes, woven and nonwoven support materials, wovenand nonwoven filtration media, barrier materials, and other materials.The sample collection patch may further include additional substances,such as reagents, buffers, probes, surfactants, binders, indicators,preservatives, and the like, such as may be useful in performing variousassays.

[0059] Such filtration media may be bonded to a flexible structure,spring or other support to allow movement in and out of the collectionchamber and to also allow ease of removal of the material for samplehandling and removal. Alternatively, a closed-cell foam structure may besubstituted for such media, thereby allowing movement in the chamberwhile maintaining contact with the nipple, either as a stand-alonecollection medium or with a membrane bonded at the surface of themedium. Such media may also act as contamination barriers, protectingthe powerhead circuit and pump system in the control console fromcontamination by body fluids in the negative pressure air path.

[0060] Thus, the present invention provides a method of screeningintraductal breast fluid for one or more breast disease markers. Themethod comprises the steps of contacting the breast with a mechanicalintraductal fluid aspiration device, and activating the device to applyperistaltic compression and suction to the breast during a period ofnonlactation to remove intraductal breast fluid. The fluid is thereafterscreened such as by cytological examination and/or biochemical screeningfor breast disease markers. In one embodiment, the method furthercomprises the step of applying heat from the device to the breast.

[0061] There is also provided an intraductal breast fluid screeningdevice. The device comprises a tissue contacting surface defining afirst concavity for receiving a breast and a second concavity forreceiving a nipple. A driver for imparting compressive force on at leasta portion of the tissue contacting surface defining the first concavityis additionally provided. A vacuum conduit is provided in communicationwith the second concavity, and a sample collector is provided incommunication with the second concavity. The sample collector may be areservoir, or an absorptive patch for absorbing or retaining a sample.Preferably, the collection patch is removable from the aspirationdevice.

[0062] The required sample size may vary, depending upon the intendedassay or screening test. For example, relatively larger fluid volumeswill be required for cytological examination as is well understood inthe art. Relatively smaller sample sizes may suffice for monoclonalantibody or other specific binding chemistry assays or biochemicalmarkers.

[0063] Although the present inventors believe that sufficient samplevolume will be obtainable from most patients using the heat, compressionand suction cycles provided by the pump disclosed herein, some patientsmay benefit from administration of one or more agents to enhanceproductivity. For example, oxytocin may be administered, preferably viaintranasal administration, in amounts effective to stimulate mammaryfluid expression in the patient. Once a sufficient post-administrationtime period has elapsed to allow the oxytocin to reach and stimulatetarget alveolar-ductal tissues, the breast is pumped and a biologicalsample is collected, as described above. After the sample is collected,a bioassay is conducted on the sample to determine the presence and/oramount of a selected breast disease marker, preferably a breast cancermarker or panel of breast cancer markers, in the sample.

[0064] One additional manner of increasing the collected fluid volume isto introduce a carrier fluid retrograde into the duct, such as throughthe use of a pressurized stream directed to the external opening of theduct. The carrier may alternatively be introduced using an introductionneedle or cannula which is advanced either transluminally through theduct or percutaneously. The carrier fluid may increase mobilization ofcellular fragments and other markers, which will be available uponaspiration of the fluid for assay. Aspiration may occur eitherimmediately following introduction of the carrier fluid, or after asufficient indwelling period of time to permit mobilization of carriersoluble or carrier transportable cells, cell components, or markers.

[0065] Any of a wide variety of carriers may be utilized, depending uponthe desired clinical objective. For example, an aqueous solution may beprovided with any of a variety of drugs or other active agents to eithertreat the breast, or facilitate the release and/or transport ofidentifiable markers.

[0066] Thus, there is provided in accordance with the present inventiona method of screening for breast cancer or other breast disease,comprising the steps of providing a patient having at least one breastduct with an external opening. A stream of carrier fluid is directedunder pressure into the opening to introduce a volume of carrier fluidinto the duct. The fluid is thereafter removed from the duct through theexternal opening, and the removed carrier fluid is screened for at leastone indicium of a physiological condition such as a marker as discussedin greater detail elsewhere herein. The removing carrier fluid step ispreferably accomplished by the application of suction to the externalopening of the duct. Preferably, suction is accompanied by compressionsuch as peristaltic or other systemic compression. The compressiondevice is preferably heated, such as in accordance with the devicediscussed above. The screening step may be accomplished by screening forcytologically abnormal cells, or markers as discussed in detailelsewhere herein.

[0067] Another aspect of the method includes the introduction of atherapeutic species into a breast duct, with or without subsequentaspiration for marker assay. In accordance with this method, a media isprovided, comprising a carrier and at least one therapeutic species. Astream of a media is directed at the external opening to the duct, tointroduce media into the duct.

[0068] Any of a variety of devices may be utilized, to direct apressurized fluid stream. See, for example, U.S. Pat. No. 5,399,163 toPeterson, et al., entitled “Needleless Hypodermic Injection Methods andDevice,” the disclosure of which is incorporated in its entirety hereinby reference. Such devices are currently known in the arts of needlelessinjection and surgical pressurized water cutting devices, both of whichmay be modified to reduce the velocity of the fluid stream so that it isinsufficient to cause tissue damage but sufficient to introduce carrierfluid retrograde into the duct. Introduction may be further facilitatedby optimizing the viscosity and temperature of the fluid carrier, whichmay be accomplished through routine experimentation by those of ordinaryskill in the art in view of the disclosure herein. Powered carrierintroduction is preferably preceded by keratin plug removal, asdiscussed elsewhere herein.

[0069] As used herein, the term breast disease marker refers to anycell, cell fragment, protein, peptide, glycoprotein, lipid, glycolipid,proteolipid, or other molecular or biological material that is uniquelyexpressed (e.g. as a cell surface or secreted protein) by diseasedbreast cells, or is expressed at a statistically significant, measurablyincreased or decreased level by diseased breast cells, or in associationwith breast disease (e.g. a protein expressed by an infectious agentassociated with breast disease), or is expressed at a statisticallysignificant, measurably increased or decreased level by diseased breastcells compared to normal breast cells, or which is expressed bynon-diseased breast cells in association with breast disease (e.g. inresponse to the presence of diseased breast cells or substances producedtherefrom). Breast disease markers can also include specific DNA or RNAsequences marking a deleterious genetic change, or an alteration inpatterns or levels of gene expression significantly associated withbreast disease. Preferred breast disease markers include markers ofbreast infections, benign neoplasia, malignant neoplasia, pre-cancerousconditions, and conditions associated with an increased risk of cancer.Breast disease markers include breast cancer markers.

[0070] As used herein, the term breast cancer marker refers to a subsetof breast disease markers, namely any protein, peptide, glycoprotein,lipid, glycolipid, proteolipid, or other molecular or biologicalmaterial that is uniquely expressed (e.g. as a cell surface or secretedprotein) by cancerous cells, or is expressed at a statisticallysignificant, measurably increased or decreased level by cancerous cellscompared to normal cells, or which is expressed by non-cancerous cellsin association with cancer (e.g. in response to the presence ofcancerous cells or substances produced therefrom). Breast cancer markerscan also include specific DNA or RNA sequences marking a deleteriousgenetic change, or an alteration in patterns or levels of geneexpression significantly associated with cancer. In addition, breastcancer markers can include cytological features of whole cells presentin mammary fluid, such as nuclear inclusions or cytoplasmic structuresor staining attributes uniquely expressed by, or associated with,cancerous cells.

[0071] Among the breast cancer markers that are useful within themethods of the invention, a subset are described in representativereview articles by Porter-Jordan et al., Hematol. Oncol. Clin. NorthAmer. 8: 73-100, 1994; and Greiner, Pharmaceutical Tech, May, 1993, pp.28-44, each incorporated herein by reference in its entirety. Othersuitable markers are also widely known and can be readily incorporatedinto the methods of the invention using information and methodsgenerally known or available in the literature. Preferred breast cancermarkers for use within the invention include well characterized markersthat have been shown to have important value for determining prognosticand/or treatment-related variables in human female patients. As notedpreviously, prognostic variables are those variables that serve topredict outcome of disease, such as the likelihood or timing of relapseor survival. Treatment-related variables predict the likelihood ofsuccess or failure of a given therapeutic program. Determining thepresence or level of expression or activity of one or more of thesemarkers can aid in the differential diagnosis of patients with malignantand benign abnormalities, and can be useful for predicting the risk offuture relapse or the likelihood of response to a selected therapeuticoption.

[0072] It is important to note, however, that the invention does notrely solely on breast disease markers that meet the stringentrequirements of sensitivity and specificity that would render the markerimmediately acceptable for clinical application to human patients. Onthe contrary, a number of breast disease markers contemplated within theinvention fall short of these stringent criteria, and nonethelessprovide useful information that can be of substantial benefit indetecting, differentially diagnosing or managing breast health includingbreast cancer. Such non-clinically accepted markers are useful forimmediate application within the methods of the invention as basicresearch tools, and as adjunctive tools in clinical applications. Beyondthese immediate applications, many such markers are expected to befurther developed and refined according to the methods of the inventionto the point of direct clinical applicability, particularly in assaymethods that analyze combinations of markers to generate complementarydata of greater predictive value than data yielded by individual markersalone.

[0073] The preferred assay methods of the invention particularly focuson breast cancer markers associated with tumorigenesis, tumor growth,neovascularization and cancer invasion, and which by virtue of thisassociation provide important information concerning the risk, presence,status or future behavior of cancer in a patient. As noted previously,tumorigenesis and tumor growth can be assessed using a variety of cellproliferation markers (for example Ki67, cyclin D1 and PCNA). Tumorgrowth can also be evaluated using a variety of growth factor andhormone markers (for example estrogen, EGF, erbB-2, and TGF.alpha.),receptors of autocrine or exocrine growth factors and hormones (forexample IGF and EGF receptors), or angiogenic factors. In addition totumorigenic, proliferation and growth markers, a number of markersprovide information concerning cancer invasion or metastatic potentialin cancer cells, for example by indicating changes in the expression oractivity of cell adhesion or motility factors. Exemplary markers in thiscontext include Cathepsin D, plasminogen activators and collagenases. Inaddition, expression levels of several putative tumor “suppressor”genes, including nm23, p53 and rb, provide important data concerningmetastatic potential, or growth regulation of cancer cells. Assaysdirected to divalent cations, such as Ca²⁺, Zn²⁺, and the like may alsobe helpful in providing important information concerning the risk,presence, status or future behavior of breast cancer. A large number andvariety of suitable breast cancer markers in each of these classes havebeen identified, and many of these have been shown to have importantvalue for determining prognostic and/or treatment-related variablesrelating to breast cancer.

[0074] Depending upon the chemistry of any particular assay, the resultsmay be processed and expressed in a variety of ways. For example, forcertain assays, a color change may be expressed directly from the samplecollection patch in the pump. For other assays, the sample collectionpatch may be removed from the pump and developed in a desk topdeveloping kit which includes whatever reagents, rinse solutions orother materials may be necessary to produce a result. For other assays,the sample collection patch is mailed or otherwise transported to asuitable laboratory for processing.

[0075] Prior to or concurrent with each assay run of the invention,particularly in the case of assays preformed at a remote laboratory, apreliminary evaluation may be performed to verify sample origin and/orquality. The focus of such preliminary evaluations is to verify that thesample collected in the collection patch is indeed of mammary origin,and is not contaminated with other potential contaminants, such as sweatfrom skin surrounding the nipple. For these sample verificationpurposes, a variety of assays are available which identify mammary fluidmarkers known to be present in mammalian mammary fluid, and which arepreferably highly specific markers for mammary fluid (i.e. markers whichare typically always present in mammary fluid and which are absent fromall, or most of, other potentially contaminating bodily fluids andtissues).

[0076] However, an acceptable level of specificity for mammary fluidmarkers within the methods of the invention is provided by markers thatare simply known to be present in mammary fluid, even though they may bepresent in other bodily fluids. One such market is the enzyme lysozyme,which is a normal component of human serum, urine, saliva, tears, nasalsecretions, vaginal secretions, seminal fluid, and mammary fluid.Lysozyme (muramidase) is an enzyme which hydrolyzes beta 1,4-glycosidiclinkages in the mucopolysaccharide cell wall of a variety ofmicroorganisms resulting in cell lysis. Quantitative measurement oflysozyme is readily accomplished by a well known agar plate diffusionmethod, described in detail in the instructions provided with theQuantiplate.RTM. lysozyme test kit, available from Kallestad, SanofiDiagnostics (Chasta, Minn.), incorporated herein by reference in itsentirety.

[0077] Other mammary fluid markers for sample verification that are morespecific than lysozyme are preferred within the methods of theinvention, and can be readily incorporated within the invention based onpublished and generally known information. The most preferred amongthese markers are proteins and other biological substances that arespecifically expressed or enriched in mammary fluid. A diverse array ofsuitable markers in this context have been characterized and havealready been used to develop specific antibodies, including affinitypurified and monoclonal antibodies. These antibodies can in turn beemployed as immunological probes to determine the presence or absence,and/or to quantify, selected mammary fluid markers to verify mammaryfluid sample origin and quality.

[0078] Mammary fluid markers of particular interest for use within theinvention include specific cytokeratins that are characteristicallyexpressed by normal and cancerous mammary epithelial cells, againstwhich specific panels of antibody probes have already been developed.(See for example, Nagle, J. Histochem. Cytochem. 34: 869-881, 1986,incorporated herein by reference in its entirety). Also useful asmammary fluid markers are the human mammary epithelial antigens(HME-Ags) corresponding to glycoprotein components of the human milk fatglobulin (HMFG) protein, against which specific antibodies (e.g. antiHMFG1, Unipath, U.K.) are also available. (see Rosner et al., CancerInvest. 13: 573-582, 1995; Ceriani et al. Proc. Natl. Acad. Sci. USA 74:582-586, 1982; Ceriani et al., Breast Cancer Res. Treat. 15; 161-174,1990, each incorporated herein by reference in its entirety).

[0079] To conduct the breast disease marker assays provided within theinvention, a collected biological sample from mammary fluid is generallyexposed to a probe that specifically binds to a selected breast diseaseor breast cancer marker, or otherwise interacts with the marker in adetectable manner to indicate the presence or absence, or amount, of thebreast disease or breast cancer marker in the sample. Selected probesfor this purpose will generally depend on the characteristics of thebreast disease marker, i.e. on whether the marker is a proteinpolynucleotide or other substance. In preferred embodiments of theinvention, the breast disease marker is a protein, peptide orglycoprotein, all of which are effectively targeted in breast diseasemarker assays using specific immunological probes. These immunologicalprobes can be labeled with a covalently bound label to provide a signalfor detecting the probe, or can be indirectly labeled, for example by alabeled secondary antibody that binds the immunological probe to providea detectable signal.

[0080] General methods for the production of non-human antisera ormonoclonal antibodies (e.g., murine, lagormorpha, porcine, equine) arewell known and may be accomplished by, for example, immunizing an animalwith a selected breast disease marker protein, peptides synthesized toinclude part of the marker protein sequence, degradation productsincluding part of the marker protein sequence, or fusion proteinsincluding all or part of the marker protein linked to a heterologousprotein or peptide. Within various embodiments, monoclonal antibodyproducing cells are obtained from immunized animals, immortalized andscreened, or screened first for the production of an antibody that bindsto the selected breast cancer marker protein or peptide, and thenimmortalized.

[0081] It may be desirable to transfer the antigen binding regions(i.e., F(ab′)2 or hypervariable regions) of non-human antibodies intothe framework of a human antibody by recombinant DNA techniques toproduce a substantially human molecule. Methods for producing such“humanized” molecules are generally well known and described in, forexample, U.S. Pat. No. 4,816,397 (incorporated herein by reference inits entirety). Alternatively, a human monoclonal antibody or portionsthereof may be identified by first screening a human B-cell cDNA libraryfor DNA molecules that encode antibodies that specifically bind to theselected breast disease marker according to the method generally setforth by Huse et al. (Science 246: 1275-1281, 1989 (incorporated hereinby reference in its entirety). The DNA molecule may then be cloned andamplified to obtain sequences that encode the antibody (or bindingdomain) of the desired specificity.

[0082] Also contemplated within the invention are bifunctionalantibodies having independent antigen binding sites on eachimmunoglobulin molecule (as disclosed for example in Thromb. Res. Suppl.X: 83, 1990, and in The Second Annual IBC International Conference onAntibody Engineering, A. George ed., Dec. 16-18, 1991; each incorporatedherein by reference in its entirety), as well as panels of individualantibodies having differing specificities. Bifunctional antibodies andantibody panels of particular use within the invention includeantibodies and panels of antibodies that bind to two or more selectedbreast disease markers to generate complementary data of greaterpredictive value than data yielded by individual markers alone.

[0083] Monoclonal antibodies are particularly useful within theinvention as labeled probes to detect, image and/or quantify thepresence or activity of selected breast disease markers. In thiscontext, monoclonal antibodies that specifically bind to selected breastdisease markers are provided which incorporate one or more well knownlabels, such as a dye, fluorescent tag or radiolabel. By incorporatingsuch a label, the antibodies can be employed in routine assays todetermine expression, localization and/or activity of one or moreselected breast disease markers in a biological sample including, orderived from, mammary fluid.

[0084] Results of these assays to determine expression, localizationand/or activity of a selected breast disease marker in a test sampletaken from a patient at risk for breast disease, or known to have breastdisease, can be compared to results from control studies detectingand/or quantifying the same marker in biological samples obtained fromnormal patients negative for breast disease. In this manner, baselinedata and cutoff values can be determined according to routine methods torefine the assays of the invention and adapt them for direct clinicalapplication.

[0085] Detection and/or quantification of breast disease markers in thebiological samples of the invention can be accomplished using a varietyof methods. Preferred methods in this regard include well known ELISAimmunoassays, immunoprecipitation assays, and various solid phaseimmunoassays including Western blotting, dot blotting and affinitypurification immunoassays, among other methods. Comparable methods aredisclosed herein, or are elsewhere disclosed and known in the art, forusing non-antibody probes to detect and/or quantify the expressionand/or activity of breast disease markers. Suitable non-antibody probesfor use within the invention include, for example, labeled nucleotideprobes that hybridize at standard or high stringency to DNA transcriptsof oncogenes and other DNA sequences associated with elevated breastdisease risk, or with mRNA transcripts encoding breast disease markerproteins. Other suitable probes include labeled ligands, bindingpartners and co-factors of breast disease markers (e.g. growth factorreceptor ligands, or substrates of breast cancer associated proteasessuch as Cathepsin D).

[0086] In certain embodiments of the invention, cDNA and oligonucleotideprobes are employed in Northern, Southern and dot-blot assays foridentifying and quantifying the level of expression of a selected breastdisease marker in cell samples collected from expressed mammary fluid.Measuring the level of expression of breast disease markers according tothese methods will provide important prognostic and treatment-relatedinformation for assessing a broad range of breast disease, including thegenesis, growth and invasiveness of cancer, in mammals, particularlyhumans. For example, assays utilizing oligonucleotide probes will assistearly screening to evaluate heritable genetic lesions associated withbreast cancer, and to distinguish between pre-cancerous, early cancerousand likely metastatic lesions in patients.

[0087] In addition to the above mentioned sample extraction, collectionand assay methods, the invention also provides kits and multicontainerunits comprising reagents and components for practicing the samplecollection and assay methods of the invention. Briefly, these kitsinclude basic components for obtaining a biological sample from mammaryfluid.

[0088] A pharmaceutical preparation of oxytocin in a biologicallysuitable carrier may optionally be included. Preferably, the oxytocinpreparation is provided in an intranasal spray applicator and containsapproximately 40 USP units of oxytocin per ml of liquid carrier, whichcarrier is a simple, inexpensive buffered saline solution. Preferredapplicators can be in any of a variety of pressurized aerosol orhand-pump reservoir forms, with a nozzle for directing a liquid spray ofthe oxytocin into a patient's nostril.

[0089] The breast pump of the present invention is also provided. Thepump is designed to generate intermittent or sustained negativepressures in an area surrounding the nipple of between about 50-200 mmHg, as well as heat and compression as has been discussed. Preferably,the breast pump serves a dual purpose of facilitating mammary fluidexpression from the nipple, and to provide the reservoir or solid phasecollecting device incorporated within the breast pump for biologicalsample collection.

[0090] Kits for practicing the assay methods of the invention include asuitable container or patch or other device for collecting a biologicalsample from expressed mammary fluid. A range of suitable collectiondevices are contemplated corresponding to a wide range of suitablebiological samples that may be collected from the expressed mammaryfluid. For example, simple sterile containers or reservoirs are providedto collect whole mammary fluid. Alternatively, a variety of solid phasedevices, including glass or plastic slides, membranes, filters, beadsand like media, are provided to receive or partition selected liquid orsolid fractions of the mammary fluid, to receive or partition cells orcellular constituents from the mammary fluid, or to receive or partitionpurified or bulk proteins, glycoproteins, peptides, nucleotides(including DNA and RNA polynucleotides) or other like biochemical andmolecular constituents from the mammary fluid. A wide variety of suchsample collection devices can be readily adapted for use within specificembodiments of the invention. These collection devices may be providedas a component of the breast pump (such as a removable fluid reservoiror nitrocellulose filter placed within the pump to directly receive orcontact the expressed mammary fluid as it is pumped), or may be providedseparately (for example as a non-integral membrane, filter, affinitycolumn or blotting material to which mammary fluid or mammary fluidcomponents are exposed to collect a biological sample for assaypurposes).

[0091] Although the foregoing invention has been described in terms ofcertain preferred embodiments, other embodiments and applications willbecome apparent to those of ordinary skill in the art in view of thedisclosure herein. Accordingly, the present invention is not intended tobe limited by the recitation of preferred embodiments, but is intendedto be defined solely by reference to the appended claims.

What is claimed is:
 1. A method of screening intraductal breast fluidfor one or more breast disease marker, comprising the steps ofcontacting the breast with a mechanical intraductal fluid aspirationdevice, activating the device to apply peristaltic compression andsuction to the breast during a period of non lactation to removeintraductal breast fluid, and screening the fluid for breast diseasemarkers.
 2. A method of screening intraductal breast fluid as in claim1, further comprising the step of applying heat from the device to thebreast.
 3. An intraductal breast fluid screening device, comprising: atissue contacting surface defining a first concavity for receiving abreast and a second concavity for receiving a nipple; a driver, forimparting compressive force on at least a portion of the tissuecontacting surface defining the first concavity; a vacuum conduit incommunication with the second concavity; and a sample collector incommunication with the second concavity.
 4. An intraductal breast fluidaspiration device as in claim 3, wherein the driver imparts peristalticcompressive force on the tissue contacting surface.
 5. An intraductalbreast fluid aspiration device as in claim 3, further comprising a heatsource thermally coupled to the tissue contacting surface.
 6. Anintraductal breast fluid aspiration device as in claim 3, wherein thedriver comprises a motor.
 7. An intraductal breast fluid aspirationdevice as in claim 3, wherein the driver comprises at least oneexpandable chamber.
 8. An intraductal breast fluid aspiration device asin claim 7, wherein the chamber is defined within a flexible tube.
 9. Anintraductal breast fluid aspiration device as in claim 3, furthercomprising a vacuum source in communication with the vacuum conduit. 10.An intraductal breast fluid aspiration device as in claim 3, wherein thesample collector comprises a collection patch.
 11. An intraductal breastfluid aspiration device as in claim 3, wherein the collector isremovable from the aspiration device.
 12. An intraductal breast fluidaspiration device as in claim 10, wherein the sample collection patchcomprises a binding system for binding at least one analyte of interestin the breast fluid.
 13. An intraductal breast fluid aspiration deviceas in claim 12, wherein the binding system comprises a monoclonalantibody.
 14. An intraductal breast fluid aspiration device as in claim3, further comprising a microprocessor for controlling the driver. 15.An intraductal breast fluid aspiration device as in claim 3, furthercomprising a housing, wherein the tissue contacting surface is removablycarried by the housing.
 16. A portable, self contained, intraductalfluid screening device, comprising: a housing; a breast interface on thehousing; at least one cell and cell fragment collector in communicationwith the breast interface; a vacuum source in communication with theinterface; a compression driver coupled to the interface; and at leastone control on the housing for controlling operation of the aspirationdevice.
 17. A portable, self contained, intraductal fluid aspirationdevice as in claim 16, wherein the interface is removably connected tothe housing.
 18. A portable, self contained, intraductal fluidaspiration device as in claim 16, comprising a fluid reservoir incommunication with the interface.
 19. A portable, self contained,intraductal fluid aspiration device as in claim 18, wherein the fluidreservoir is removably attached to the housing.
 20. A portable, selfcontained, intraductal fluid aspiration device as in claim 16, furthercomprising a heating element in thermal communication with theinterface.
 21. A portable, self contained, intraductal fluid aspirationdevice as in claim 16, further comprising an ultrasonic transducer incommunication with the interface.
 22. An intraductal fluid aspirationdevice, comprising: a control unit; a power head; a flexible controlline connecting the power head to the control unit; a disposable userinterface removably attached to the power head; a vacuum source in thecontrol unit, in communication with the user interface through thecontrol line; a heat source in thermal communication with the userinterface; and a compression cycle generator in force transmittingcontact with the user interface.
 23. An intraductal fluid aspirationdevice as in claim 22, further comprising a central processing unit inthe control unit for controlling the delivery of heat, compression andsuction through the user interface.
 24. A method of increasing yield ina breast ductal aspiration, comprising the steps of: providing acarrier; introducing the carrier under pressure retrograde into theduct; and recovering the carrier from the duct, using compression, heatand suction.
 25. A method as in claim 24, wherein the introducing thecarrier step comprises directing a stream of the carrier at the externalopening of the duct.
 26. A method as in claim 24, wherein the recoveringthe carrier step is assisted by administering oxytocin.
 27. A method asin claim 24, further comprising the step of clearing the duct in thevicinity of the external opening to facilitate fluid flow, prior to theintroducing the carrier step.
 28. A method of enhancing the transport ofintraductal indicium of a physiological condition for diagnosticanalysis, comprising the steps of: transductally introducing a carrierunder pressure retrograde into a breast duct; and retrieving the carrierfrom the duct for diagnostic analysis.
 29. A method as in claim 28,wherein the carrier comprises a component for enhancing transport of theindicium from the duct.
 30. A method as in claim 28, wherein theindicium comprises a metabolite.
 31. A method as in claim 28, whereinthe indicium comprises carcinomatous or dysplastic cells.
 32. A methodas in claim 28, wherein the retrieving the carrier step comprisesaspirating the carrier under suction and compression.
 33. A method ofscreening for breast cancer in a patient, comprising the steps of:providing a patient having at least one breast duct with an externalopening thereon; directing a stream of carrier fluid under pressure intothe opening to introduce a volume of carrier fluid into the duct;removing carrier fluid from the duct through the external opening; andscreening the removed carrier fluid for at least one indicium of aphysiological condition.
 34. A method as in claim 33, wherein theremoving carrier fluid step is assisted by the application of suction tothe external opening of the duct.
 35. A method as in claim 33, whereinthe screening step comprises screening for cytologically abnormal cells.36. A method of introducing a therapeutic species into a breast duct,comprising the steps of: identifying a patient having at least onebreast duct with an external opening thereon; providing a mediacomprising a carrier and at least one therapeutic species; directing astream of the media at the external opening to the duct to introduce themedia into the duct.
 37. A method as in claim 36, wherein the carriercomprises a liquid.
 38. A method as in claim 36, further comprising thestep of manipulating the duct to enhance transport of the media withinthe duct.
 39. A method as in claim 36, further comprising the step ofapplying suction to the external opening to remove media from the duct.40. A method as in claim 33, wherein the indicium comprises a breastdisease marker.